42,000-molecular weight EGF receptor has protein kinase activity

The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor c(. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 6 1 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent V,,, value of 20 nmol min-' mg-' and K , values of 4.5 pM for ATP and 1.43 mM for angiotensin 11. This corresponds to a turnover number of 1.4 mol min-' mol-' .Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin I1 as substrate. The specific activity of the intracellular domain of the EGF-R, when assayed at 20°C in the presence of 1M ammonium sulfate, was 160 nmol min-' mg-'. Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific. No activation was found when assayed using polymeric substrates. Addition of MeZ+-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDSjPAGE migration. Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2: 5 : 6 : l), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGF-R. Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme.The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to epidermal growth factor (EGF) and transforming growth factor a. The receptor consists of an extracellular hormone-binding domain, a short hydrophobic transmembrane domain and an intracellular domain which contains the protein-tyrosine kinase sequence (Carpenter, 1987; Yarden and Ullrich, 3988a). Enzymatic activity of the kinase domain is essential for signal transduction via the EGFCorrespondence to N. B. Lydon,

Section: Discussionmentioning

confidence: 99%

Large‐scale purification and characterisation of a recombinant epidermal growth‐factor receptor protein‐tyrosine kinase

McGlynn

Becker

Mett

et al. 1992

“…The synthesis of a functional gene product was dcmonstrated by means of a protein kinase assay. This confirins the previous observation [3] that this region of the EGF receptor retains catalytic activity even after deletion of the EGF-binding region, transmembrane domain and autophosphorylation sites, and also shows that it is possible to express active human EGF-receptor protcin kinase domain protein in E. coli.…”

Section: U1-u4mentioning

confidence: 60%

“…Based on the complete amino acid sequence deduced from the nucleotide sequences of cDNA clones [2], it was shown that the receptor consists of an extracellular EGF-binding domain, a hydrophobic transmembrane segment, a cytoplasmic domain which has a catalytic portion possessing tyrosine kinase activity [3] and several autophosphorylation sites. The kinase can mediate autophosphorylation, phosphorylate a variety of other proteins and is activated by EGF-binding.…”

mentioning

confidence: 99%

Synthesis of a gene for the protein kinase domain of the epidermal growth factor receptor and its expression in Escherichia coli

Farrow

Kamiya

Miura

et al. 1989

A gene encoding the protein kinase domain of the epidermal growth factor receptor has been chemically synthesised, cloned and expressed in Escherichia coli. The 942-base-pair gene was constructed by enzymatic ligation of 56 oligonucleotides and cloned into an expression vector downstream of the E. coli trp promoter.Production of active gene product was confirmed by means of a protein kinase assay, demonstrating that the enzymatic activity of the protein kinase domain of the epidermal growth factor receptor is retained after expression in E. coli.A number of the cellular DNAs with sequences similar to transforming genes have been shown to encode growth factors or growth factor receptors, which when either altered or constitutively expressed may cause loss of normal growth control. The receptor for epidermal growth factor (EGF) has extensive similarity with the v-erbB protein, and the neu(cerbB2,HER2) oncogene product is also structurally similar to the EGF receptor and v-erbB protein and is likely to be another transmembrane receptor for an as yet unidentified ligand. Transforming growth factor CI is highly related to EGF, binds to the EGF receptor with similar affinity and induces proliferation of cells bearing the EGF receptor (for a review see [l]).The human EGF receptor is a 170-kDa transmembrane glycoprotein which mediates the mitogenic response of target cells to EGF. Based on the complete amino acid sequence deduced from the nucleotide sequences of cDNA clones [2], it was shown that the receptor consists of an extracellular EGF-binding domain, a hydrophobic transmembrane segment, a cytoplasmic domain which has a catalytic portion possessing tyrosine kinase activity [3] and several autophosphorylation sites. The kinase can mediate autophosphorylation, phosphorylate a variety of other proteins and is activated by EGF-binding. The v-erbB protein is a truncated form of the EGF receptor, lacking most of the EGF-binding domain and the C-terminal portion containing the autophosphorylation sites [l]. Mutagenesis of the tyrosine kinase domain has shown that kinase-negative mutants are defective in the transduction of the mitogenic effect of EGF.

“…Tyr992, known to associate with phospholipase Cy, is a likely phosphorylation site candidate, though the phosphorylated residue in the 42-kDa kinase has not been identified. A similar, catalytically active, 42-kDa fragment has been isolated in v i m by tryptic digestion of intact EGF receptor (Basu et al, 1984;Koland and Cerione, 1990). Although the N-terminus of the tryptic fragment has not been identified, it has been suggested to be C-terminal to Thr6.54 (Koland and Cerione, 1990).…”

Section: Discussionmentioning

confidence: 99%

“…Accessibility Of this region to several proteases suggested that it is relatively exposed (Basu et al, 1984;Hunter, 1984;Gates and King, 198.5 …”

Section: Discussionmentioning

confidence: 99%

The calpain cleavage sites in the epidermal growth factor receptor kinase domain

Gregoriou

Willis

Pearson

et al. 1994

The proteolysis of the human epidermal growth factor receptor cytoplasmic domain by calpain has been studied in vitro using purified recombinant cytoplasmic domain expressed in insect cells. Limited proteolysis produced kinase that was truncated at either N‐ or C‐termini, as well as in the hinge region. We identified seven sites of calpain proteolysis by N‐terminal sequencing of purified fragments. Calpain cleaved between the catalytic and autophosphorylation domains at two sites in the sequence Gln996–Asp1059, in the hinge region. Three new sites were also found in the autophosphorylation domain, preceding each of the major autophosphorylation sites. A fourth new site was located in the juxtamembrane domain, C‐terminal to the regulatory Thr654. We purified an active 42‐kDa fragment generated by calpain proteolysis between Leu659–Gln660 in the juxtamembrane domain, and in the hinge region. A fifth new site of calpain cleavage was found between the nucleotide binding motif Gly‐Xaa‐Gly‐Xaa‐Xaa‐Gly and the essential Lys721 in the catalytic core of the kinase. Since both of these features are required for catalysis, calpain cleavage at this site may potentially provide a mechanism for down‐regulation of kinase activity in vivo, under conditions of calpain activation. Thus the distribution of calpain cleavage sites along the kinase domain is consistent with a role for calpain both as a processing and as a degradative protease in epidermal growth factor receptor signalling.